Chromium (VI)-resistant strain of Shewanella alga

ABSTRACT

A chromium-resistant strain of Shewanella alga BrY and method of using such for removing chromium (VI) from a chromium-contaminated waste stream involving treating the waste stream with the chromium-resistant strain under anaerobic conditions whereby Cr(VI) in the waste stream is reduced, the chromium generally forming a precipitate which can be separated from the waste stream.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. Ser. No. 08/368,454,filed Jan. 4, 1995, entitled "METHOD FOR BACTERIAL REDUCTION OF CHROMIUM(VI)".

BACKGROUND

The present invention relates to a method for reducing chromium (VI) andmore particularly to a method using a chromium (VI)-resistantdissimilatory bacterial strain to reduce chromium (VI).

Hexavalent chromium (Cr(VI)) is a common toxic heavy metal pollutant inwastewaters and soils and elsewhere in natural ecosystems. Wastewaterscontaining Cr(VI) are produced by many industrial processes includingchromium plating, metal cleaning and processing, wood preparation andalloy preparation. These wastewaters must be treated before beingdischarged into the environment. Chromium is also used in production ofpigments, tanned leathers, fungicides, corrosion inhibitors in coolingwater and drilling muds, wall paper, photographic films, magnetic tapes,printing inks and as a catalyst in the synthesis of many organicchemicals. Leakage, poor storage and improper disposal practices havereleased chromium into the environment in numerous locations causingcontamination of ground and surface waters and of soils. Thesechromium-contaminated sites are subject to remediation requirements toreduce or eliminate the toxic levels of chromium.

As a result, efficient methods for cleaning up waste streams and forremedial clean up of contaminated sites are of critical importance.Conventional methods for removing toxic Cr(VI) ions include chemicalreduction followed by precipitation, ion exchange and adsorption on coalactivated carbon, alum, kaolinite, and fly ash. Most of these methodsrequire high levels of energy or large quantities of chemical reagents.Other remediation methods involve the use of microorganisms such as thestrain HO1 of Enterobacter cloacae which can anaerobically reduce Cr(VI)(Komori, K.; Kiyoshi, T.; and Hisao, O.; "Effects of Oxygen Stress onChromate Reduction in Enterobacter cloacae Strain HO1", 1990, Journal ofFermentation and Bioengineering, 69(1):67-69). The Enterobacter strainHO1 can reduce Cr(VI) at levels of around 1-2 mM of potassium chromate,but levels above 5 mM are lethal to the bacteria. Some strains ofPseudomonas and Aeromonas which are capable of reducing Cr(VI) have alsobeen identified.

Although some bacteria apparently use Cr(VI) as a terminal electronacceptor, it is not apparent that Cr(VI) reduction yields sufficientenergy to support their anaerobic growth and reproduction. Althoughorganisms such as Pseudomonas chromatophila (Lebedeva, E. V. andLyalikova, N. N. "Reduction of crocoite by Pseudomonas chromatophila sp.nov.", 1979, Microbiology 48:517-522), Pseudomonas fluorescens (Bopp, L.H. and Erlich, H. L. "Chromate resistance and reduction in Pseudomonasfluorescens strain LB300", 1988, Arch. Microbiol. 150:426-431), andEnterobacter cloacae strain HO1 (Ohtake, H., Fujii, E., and Toda, K."Bacterial reduction of hexavalent chromium: Kinetic aspects of chromatereduction by Enterobacter cloacae HO1", 1990, Biocatalysis 4:227-235;and Wang, P., Mori, T., Komori, K., Sasatsu, M., Toda, K. and Ohtake, H."Isolation and characterization of an Enterobacter cloacae strain thatreduces hexavalent chromium under anaerobic conditions", 1989, Appln.Environ. Microbiol. 55:1665-1669), can reduce Cr(VI) anaerobically, noevidence for Cr(VI)-dependent growth has been presented (Lovley, D. R."Dissimilatory metal reduction", 1993, Ann. Rev. Microbiol. 47:263-291).

The application of Cr(VI)-reducing bacteria such as Enterobacter cloacaeand Desulfovibrio vulgaris has been proposed as a potential means oftreating Cr(VI)-containing waters and waste streams. However, D.vulgaris cannot grow with Cr(VI) as an electron acceptor, and thustreatment systems using this organism would require continualreinoculation. Further, E. cloacae requires a rich, expensive,heterotrophic medium in order to reduce Cr(VI), limiting thecost-effectiveness of its use in Cr(VI) treatment systems.

Shewanella alga strain BrY is an obligately respiratory, facultativelyanaerobic bacterium which can grow anaerobically by coupling theoxidation of organic acids or H₂ to the reduction of Fe(III), Mn(IV),U(VI), (Caccavo, Jr. F., R. P. Blakemore, R. P. and Lovley, D. R. "Ahydrogen-oxidizing Fe(III)-reducing microorganism from the Great BayEstuary, New Hampshire ", 1992, Appl. Environ. Microbiol. 58:3211-3216),or Co(III)-EDTA. Further, strain BrY contains an electron transportchain and terminal reductase which can couple the oxidation of lactateor H₂ to the reduction of Cr(VI) as well. However, all attempts to growthis organism with Cr(VI) as the sole terminal electron acceptor haveheretofore been unsuccessful. Similar results have been observed withDesulfovibrio vulgaris (Lovley, D. R. and Phillips, E. J. P. "Reductionof chromate by Desulfovibrio vulgaris and its c3 cytochrome", 1993,Appl. Environ. Microbiol. 60:726-728).

Identification of a bacterial strain not only resistant to high levelsof Cr(VI) but which could also use Cr(VI) as a terminal electronacceptor in a process yielding sufficient energy to sustain continuousgrowth in situ would be a valuable means of purifying Cr(VI)-containingsoils, waters, and waste streams.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows growth of wild-type strain BrY under varying concentrationsof Cr(VI) under aerobic conditions.

FIG. 2 shows growth of strain BrY-MT under varying concentrations ofCr(VI) under aerobic conditions.

FIG. 3 shows the lack of capability of strain BrY-MT to reduce Cr(VI)under aerobic conditions.

FIG. 4 shows the anaerobic growth of strain BrY-MT using lactate as anelectron donor.

FIG. 5 shows aerobic growth of strain BrY with 100 μM Cr(VI) and SO₄ ²⁻or djenkolic acid.

FIG. 6 shows aerobic growth of strain BrY-MT with 3 mM Cr(VI) and SO₄ ²⁻or djenkolic acid.

FIG. 7 shows difference spectra of cell-free extracts of strains BrY andBrY-MT.

FIG. 8 shows a containment berm-type treatment system.

FIG. 9 shows a slurry reactor-type treatment system.

FIG. 10 shows a groundwater pumping type treatment system.

FIG. 11 shows a packed-bed type treatment tank.

FIG. 12 shows a fluidized-bed type treatment tank.

DESCRIPTION OF THE INVENTION

Identified herein is Cr(VI)-resistant mutant strain of BrY which cangain energy to support anaerobic growth by Cr(VI) reduction. TheCr(VI)-resistant mutant can be used in a variety of methods forremediating Cr(VI)-contaminated soils, waters or waste streams. When thechromium-reducing microorganisms described herein are placed in waters,soils or other waste streams containing dissolved Cr(VI), the Cr(VI) isreduced to Cr(III) which forms a precipitate which can then be removed.

In one version, the method of the present invention can be carried outin a bioreactor comprising any variety of culture vessels in which theorganisms are attached to a stationary substrate (e.g., a packed-bed) orare free-floating or are attached to free-floating substrates (e.g., afluidized-bed). If necessary, an electron donor and/or nutrient mediumare added to the reactor. The chromium-containing water is introducedinto the bioreactor wherein the Cr(VI) is reduced and forms aprecipitate which can be more easily concentrated and removed from thesystem, for example, by filtration.

In another version, the chromium-reducing bacteria may be disposed in acontainer compartment where they are separated fromchromium-contaminated water disposed therein by a semipermeablemembrane. The chromium (VI) in the water can diffuse across the membraneand into the compartment containing the bacteria where the Cr(VI) isreduced and precipitated out. In another embodiment, the bacteria may beattached to a support material within a column through whichcontaminated water can pass and where chromium (VI) can be reduced whenthe Cr(VI)-contaminated water is passed into the column.

It is also contemplated that the BrY-MT strain disclosed herein can beused to treat chromium-contaminated ground water in situ by injectingthe microorganisms, along with a suitable electron donor if necessary,into a specific "injection zone" of the subsurface. As groundwaterpasses through the injection zone, further movement of chromium isinhibited by the precipitation of Cr(VI) effected by the bacteria. Inanother version, the bacteria could be injected into the injection zonewithin semipermeable membrane containers. After a period of time inwhich the microorganisms reacted with the chromium in the groundwater,the semi-permeable containers could be removed from the injection zone,thereby removing reduced chromium from the subsurface zone.

It is also contemplated that the strain of Shewanella alga describedherein may be used to treat waste streams contaminated with Fe(III),Mn(IV), and U(VI) for the purpose of reducing these forms to a loweroxidation state.

More particularly, the present invention contemplates a method ofreducing Cr(VI) concentration in a waste stream under anaerobicconditions. In one version the invention comprises the steps ofproviding a culture of a Cr(VI)-resistant mutant strain of a firstShewanella alga strain, and treating the waste stream with the mutantstrain under anaerobic conditions wherein at least a portion of theCr(VI) in the waste stream is reduced by the mutant strain forming achromium precipitate. The method may comprise the additional step ofseparating the chromium precipitate from the waste stream.

The culture may be provided in a compartment having a semipermeablemembrane accessible to the waste stream wherein the Cr(VI) is able topass from the waste stream through the semipermeable membrane to theculture. The method may further comprise the step of adding an electrondonor to the culture. The electron donor may be selected from the groupconsisting of hydrogen, lactate, formate, and pyruvate. The culture maybe attached to a solid substrate in a container wherein the waste streamis exposed to the culture by passing the waste stream into thecontainer. The solid substrate may be a packed bed or a fluidized bed.In the step of providing a mutant strain, the mutant may be the strainhaving ATCC accession number 55627. In the step of providing a mutantstrain, the first Shewanella alga strain may be strain BrY having ATCCaccession number 51181. In the step of providing a mutant strain, themutant strain may be obtained by isolating the mutant strain after aseries of sequential transfers into media having successively higherconcentrations of Cr(VI).

In another version of the present invention, the method comprises thesteps of (1) providing a Cr(VI)-resistant microorganism able to reduceCr(VI) and which can persist as a sustainable population in the presenceof Cr(VI), (2) providing a medium comprising an electron-donorutilizable by the Cr(VI)-resistant microorganism, and (3) treating thewaste stream with the microorganism and with the medium under anaerobicconditions wherein at least a portion of the Cr(VI) is reduced by themicroorganisms to form a chromium precipitate. The method may comprisethe additional step of separating the chromium precipitate from thewaste stream. The waste stream may comprise a Cr(VI)-contaminated soil.When the waste stream is a soil, the Cr(VI)-contaminated soil may bedeposited within a chamber having the microorganism therein and whereinthe temperature of the soil within the chamber is held to within a rangeof from 20° C. to about 50° C. The electron donor may be selected fromthe group consisting of hydrogen, lactate, formate, and pyruvate.

The invention may further comprise a chromium (VI)-resistant strain ofthe Shewanella alga strain deposited under ATCC accession number 51181.The invention may further comprise a chromium (VI)-resistant strain ofShewanella alga deposited under ATCC accession number 55627. Theinvention may further comprise a biologically pure culture of Shewanellaalga strain BrY-MT deposited under ATCC accession number 55627. Theinvention may further comprise a biologically pure culture of a strainof Shewanella alga BrY which is resistant to Cr(VI) and can grow agenerally sustainable population under anaerobic conditions in thepresence of Cr(VI). The invention may further comprise a biologicallypure culture of a strain of Shewanella alga BrY which is resistant toCr(VI) up to a concentration of at least about 8 mM. The invention mayfurther comprise a biologically pure culture of a strain of Shewanellaalga BrY which is resistant to Cr(VI) up to a concentration of at leastabout 8 mM and which can grow anaerobically with Cr(VI) as the terminalelectron acceptor.

MATERIALS AND METHODS

Bacterial strains.

The source strain was a wild type strain of Shewanella alga designatedas strain BrY (ATCC accession number 51181). The Cr(VI)-resistant mutantof Shewanella alga identified herein is designated as strain BrY-MT andwas deposited with The American Type Culture Collection (ATCC) locatedat 12301 Parklawn Dr., Rockville, Md., 20852, on Oct. 20, 1994, and hasthe ATCC accession number 55627.

Media.

Media used for aerobic growth of the bacteria contained in g/L d-H₂O:Na₂ HPO₄, 2.1:KH₂ PO₄, 1.6:NH₄ Cl, 1.5; tryptic soy broth, 1.0.Anaerobic growth medium contained in g/L d-H₂ O: NaHCO₃, 2.5; KH₂ PO₄,0.6; NH₄ Cl, 1.5; tryptic soy broth, 1.0; vitamins, 10 ml, minerals, 10ml. Both aerobic and anaerobic media contained 20 mM lactate as theelectron donor. Cr(VI) was added from a sterile stock solution ofpotassium chromate. Anaerobic medium was made using standard anaerobictechniques described by Caccavo, F, Jr., McInerney, M. J., Davis, M.,Stolz, J. F., Lonergan, D. J., and Lovley, D. R. ("Geobactersulfurreducens sp. nov., a Hydrogen- and Acetate-Oxidizing DissimilatoryMetal-Reducing Microorganisms", 1994, Appl. Environ. Microbiol,60:3752-3759). Djenkolic acid and sulfate were added from sterile stocksolutions to provide a final concentration of 1.5 mM. All incubationswere at 35° C. in the dark. Aerobic cultures were shaken at 150 rpm.

Cr(VI) measurement.

Cr(VI) concentration was analyzed by the sym-diphenylcarbazide method ofUrone, P. F. ("Stability of colorimetric reagent for chromium,s-diphenycarbazide, in various solvents", 1955, Anal. Chem.27:1354-1355). Subsamples (0.5 ml) were withdrawn with a syringe andneedle, and added to 10 ml of 0.2 NH₂ SO₄. Then 0.5 ml ofs-diphenylcarbazide reagent was added. Samples were mixed, filteredthrough a 0.2 μm filter, and measured at an absorbance of 540 nm.

Cr(VI) reductase assays with washed cell suspensions were performedusing the following methods. All manipulations were performed in ananaerobic chamber. All buffers were made anaerobic by boiling andcooling under a stream of oxygen-free high purity N₂. Late-log-phasecells were harvested anaerobically by centrifugation at 7,000 X g for 20min at 4° C. and washed twice by resuspending the pellet in anaerobicPIPES Piperazine-N,N-bis(2-ethanesulfonic acid)! buffer (20 mM, pH 7.0),and recentrifuging. The final pellet was resuspended in 20 ml ofanaerobic PIPES buffer (20 mM, pH 7.0). Potassium chromate was added toBalch tubes containing 10 ml of 20 mM PIPES buffer, pH 7.0, fromanaerobic, 100 mM stock solutions in 20 mM PIPES buffer, pH 7.0, toprovide a final concentration of 500 μM. The assay tubes were sealedwith butyl rubber stoppers and aluminum crimps, the headspace wasevacuated and replaced with oxygen-free N₂, and the tubes wereautoclaved for 20 min at 115° C.

Cr(VI) reductase assays were initiated by anaerobically adding 100 μofcell extract and 10 ml of 100% H₂ to the assay tube. The assay tubeswere shaken gently and placed on their sides at 35° C. Duplicate tubeswere used for each analysis. Replicates did not differ from the mean bymore than 10%. Negative controls did not contain H₂. Cr(VI) reductionactivity assays were performed using fixed time point analyses. Cr(VI)reduction rates were linear with time for up to 5 minutes and wereproportional to protein concentration.

Growth measurements.

Aerobic cell growth was measured spectrophotometrically at 601 nm.Anaerobic growth was determined by direct cell counts using amodification of the epifluorescent microscopy technique of Hobie, J. E.,Dailey, R. J., and Jasper, S. ("Use of nucleopore filters for countingbacteria by fluorescence microscopy", 1977, Appl. Environ. Microbiol.33:1225-1228), as described by Lovley, D. R. and Phillips, E. P. J.("Novel mode of microbial energy metabolism: organic carbon oxidationcoupled to dissimilatory reduction of iron or manganese", 1988, Appl.Environ. Microbiol. 54:1472-1480).

Cytochrome analysis.

Dithionite-reduced minus air-oxidized difference spectra of cell-freeextracts were obtained using the following methods. Sodiumdithionite-reduced-minus-air-oxidized difference spectra were obtainedfor cell-free extract. Two milliliters of anaerobic, 20 mM PIPES buffer(pH 7.0) were added to 0.5 ml of cell extract in a quartz cuvette with apath length of 1.0 cm. Both the reference cuvette and sample cuvettewere air oxidized with a pipette. The sample cuvette was then reducedwith sodium dithionite. The difference spectrum was recorded with adouble beam spectrophotometer. The concentration of heme c in cell freeextracts was determined by subtracting the absorption minimum at 536 nmfrom the absorption maximum at 551 nm of thedithionite-reduced-minus-air-oxidized difference spectrum (ε⁵⁵¹⁻⁵³⁶=1.73×10 ⁴ M⁻¹ cm ⁻¹).

Chromium-Resistant Strain Isolation.

The present invention contemplates a method for culturing a bacterialstrain having at least partial resistance to a heavy metal such asFe(III), Mn(IV), U(VI) or Cr(VI) and then isolating therefrom a mutantstrain having a significantly greater resistance to the heavy metal.

In a preferred version, the present invention contemplates isolating achromium-resistant mutant from Shewanella alga strain BrY, a strainwhich has a partial resistance to chromium Cr(VI) at low concentrations.The process comprises providing a culture of Shewanella alga strain BrY.A viable population of BrY cells is then cultured in media at theminimum inhibitory concentration (MIC) of Cr(VI). The minimum inhibitoryconcentration is the highest concentration of Cr(VI) at which a viablepopulation of the BrY cells can be sustained in culture. Stated anotherway, the MIC of Cr(VI) is that concentration just below theconcentration of Cr(VI) which completely inhibits growth of strain BrY.The MIC of Shewanella alga strain BrY is approximately 250 μM. Whereused herein, the terms "Cr(VI)-resistant" cells or "chromium-resistant"cells are meant to refer to cells which are able to grow atconcentrations of Cr(VI) which are higher than the MIC of Cr(VI) forShewanella alga strain BrY, or higher than the MIC of Cr(VI) for anyother strain of Shewanella alga which exhibits at least partialresistance to Cr(VI).

After a period of growth at the MIC, a portion of the culture istransferred into another container having therein a medium with aslightly higher Cr(VI) concentration (for example, about 100 μM higher).This process is repeated whereby each succeeding culture is transferredto a medium having an incrementally-higher Cr(VI) concentration. Forexample, the increment may be 100 μM. After a predetermined number ofthese transfers into increasingly higher Cr(VI) concentrations, such asafter about 8 to 10 transfers, a strain is isolated from the finalculture medium.

The final culture-medium from which the mutant strain is removedpreferably has a Cr(VI) concentration which is at least about ten timesthat of the MIC of the original strain. In the case of BrY, the finalmedium would have a Cr(VI) concentration of at least about 2.5 to 3.0mM. Ultimately, the mutant strain which is obtained from the sequentialculturing process described herein has a MIC which is at least ten timesthat of the original strain, preferably at least twenty times, and morepreferably at least 25 to 30 times greater. Mutant strains having MICsin excess of 30 times the original MIC are most preferable.

The invention contemplates the use of any mutant strain of a Shewanellaalga which is produced by the isolation method described herein wherebythe mutant strain is isolated after a series of sequential transfersinto media having successively higher concentrations of Cr(VI).Preferably, the final medium from which the mutant strain is isolatedhas a Cr(VI) concentration which is at least about a factor of tengreater than the MIC of Cr(VI) of the parent strain of Shewanella alga.

Results

Isolation of the Cr(VI)-resistant mutant.

The Minimum Inhibitory Concentration of Cr(VI) for strain BrY wasdetermined to be approximately 250 μM. Strain BrY grew only veryslightly at 250 μM Cr(VI) under aerobic conditions (FIG. 1). Thisculture was continually transferred into an identical medium until densebacterial growth was observed. This dense culture was then sequentiallytransferred into a series of aerobic media with Cr(VI) concentrationssequentially increasing in 100 μM increments. A Cr(VI)-resistant mutant,designated herein as strain BrY-MT, was isolated from medium containing3 mM Cr(VI). This concentration was more than 10 times the MIC for thewild type strain (BrY). The MIC for strain BrY-MT was 8 mM Cr(VI) (FIG.2). Strain BrY-MT did not lose its resistance to Cr(VI) after 10transfers in aerobic medium which did not contain Cr(VI).

Cr(VI) reduction by BrY-MT.

Washed cell suspensions of both BrY and BrY-MT strains reduced Cr(VI)with lactate as the electron donor (Table I). Wild type strain BrY didnot reduce Cr(VI) under growth conditions, either aerobically oranaerobically. Strain BrY-MT did not reduce Cr(VI) under aerobic growthconditions (FIG. 3), however strain BrY-MT did grow anaerobically by theoxidation of lactate coupled to the dissimilatory reduction of Cr(VI)(FIG. 4). The increase in cell numbers coincided with the decrease inCr(VI) in the presence of lactate. Only a small amount of Cr(VI)reduction and cell growth was observed in cultures that did not containlactate. This can be attributed to a small amount of lactate transferredwith the inoculum, as cell growth and Cr(VI) reduction stopped when thislactate was depleted.

Repression/derepression assays.

Cr(VI) resistance in both BrY and BrY-MT strains depended on the sulfursource in the medium. Strain BrY grew aerobically with 100 μM Cr(VI) inthe medium (FIG. 5). When SO₄ ²⁻ was also included in the medium, growthwas enhanced. When djenkolic acid was included in the medium, growth wasinhibited. Growth of strain BrY was not inhibited by djenkolic acid inthe absence of Cr(VI), as control cultures reached an O.D. of 0.30 after12 h of incubation. Similar results were obtained when strain BrY-MT wasgrown aerobically with 3 mM Cr(VI) (FIG. 6).

Cytochrome analysis.

Difference spectra of cell-free extracts of both strain BrY and strainBrY-MT revealed absorption peaks at 551 nm, 523 nm, and 422 nm (FIG. 7).These peaks are characteristic of c-type cytochromes. Both BrY andBrY-MT contained approximately the same amount of heme c (Table 1).

                                      TABLE 1                                     __________________________________________________________________________    Cr (VI) Reductase Activity and Heme c Content of S. alga Strains                     Total Activity                                                                         Specific Activity nmoles                                                                 Total Heme c                                                                         Specific Heme c                             Strain nmoles Cr (VI)/min                                                                     Cr (VI)/min/mg protein                                                                   nmoles nmoles/mg protein                           __________________________________________________________________________    BrY-Wild Type                                                                        1288     146        35.5   1.16                                        BrY-Mutant                                                                            192      29        34     1.04                                        __________________________________________________________________________

Cr(VI)-resistant mutant.

Although the dissimilatory metal-reducing microorganism S. alga strainBrY produces an electron transport system and terminal reductase that iscapable of reducing Cr(VI), Cr(VI) appears to be toxic to the BrY strainunder growth conditions. In this study, a Cr(VI)-resistant mutant,strain BrY-MT, was isolated as described above by continually exposingthe Cr(VI)-sensitive wild type BrY to gradually increasing amounts ofCr(VI). Strain BrY-MT is resistant to Cr(VI) concentrations at levelsmore than 30 times the MIC for Cr(VI) of the BrY strain.

Cr(VI)-dependent growth of strain BrY-MT is environmentally significantbecause it demonstrates that dissimilatory metal-reducing bacteria canreduce toxic and mobile Cr(VI) to less toxic and less mobile forms whilesustaining viable Cr(VI)-reducing populations in Cr(VI) contaminatedenvironments.

Bioremediation.

These results demonstrate the S. alga strain BrY-MT can grow usingCr(VI) as a sole electron acceptor, and reduce highly toxic and mobileCr(VI) to less toxic and more immobile forms such as Cr(III) which formsa solid precipitate. Further, these results point out that in situCr(VI) bioremediation techniques using dissimilatory metal-reducingbacteria are feasible. Strain BrY-MT can grow upon and reduce Cr(VI),and thus maintain viable populations, in an inexpensive minimal medium.These attributes make strain BrY-MT the organism of choice in in vivoCr(VI) treatment systems.

Shewanella alga strain BrY-MT can be used to diminish Cr(VI)concentrations in chromium-contaminated waste streams such as soils,industrial waste, and surface and groundwaters. Chromium-contaminatedsoils can be treated ex situ for example.

One version of a soil treatment unit is shown in the treatment facilityshown in FIG. 8 designated by the general reference numeral 10. Thefacility 10 comprises a pit 12 having a liner 14 and surrounded by acontainment berm 16. A layer 18 of sand and/or gravel is disposed abovethe liner 14. A quantity of soil 20 or other solid material which iscontaminated with chromium (VI) is disposed within the pit 12 over thesand/gravel layer 18. An inoculum 22 of BrY-MT provided in a liquidmedium is then disposed over the contaminated soil 20 and percolatesthrough the soil 20. The inoculum 22 is preferably added at a rate ofabout 10⁶ -10⁸ cells per gm of soil. A drainage collection sump 24recovers runoff from the soil 20. A contamination monitoring well 26 maybe present for monitoring loss of chromium from the pit 12. Nutrientsmay be added to the inoculated contaminated soil 18 to enhance activityof the inoculum 22 although preferably sufficient nutrients are presentin the soil 20.

Shown in FIG. 9 and designated by the general reference numeral 30 isanother treatment system which can be used to treat soil 20 contaminatedwith Cr(VI). The system 30 comprises a container 32. The container 32has a mixing device 34 such as a paddle for mixing contaminated soilwhich has been inoculated with an inoculum 22 of the Cr(VI)-reducingbacterial strain. Nutrients may be added to enhance activity of theinoculum 22 and water is preferably added to form the treated soil 20into a slurry 36 which after a period of time for reaction can then bedischarged from the reaction container 32. The reacted slurry 36 willthen have a greatly reduced concentration of Cr(VI). An added benefit ofthis process is that, contaminated soil can continue to be added to thereaction container 32 as the process is ongoing. The reacted slurry 36can be collected in a container 38 for further processing or disposal.

Further the temperature and pH conditions can be closely controlled inthe container 32 to accelerate the activity of the bacteria. Preferably,the pH of the slurry 36 is maintained below 8. More preferably, the pHof the slurry 36 is maintained at about 7±0.2. The optimum temperatureof the system is 35° C.±5°, but may be in a range of from 20°-50° C.Lactate may be added as a carbon source and electron donor. Formate,pyruvate or H₂ gas may also be added as an electron donor.

Shown in FIG. 10 is a schematic of a method for treating groundwaterwhich has been contaminated by Cr(VI) in soil. The zone of contaminatedsoil 40 releases Cr(VI) to the surrounding groundwater 42. Thegroundwater 42 may be pumped via a pumping system 44 into a treatmenttank 46. The treatment tank 46 comprises a system for passing thecontaminated water 42 through a bed which sustains a live culture of theCr(VI) reducing bacteria, as explained in more detail below. The treatedwater 42 is then sent to a settling tank 48 where the reduced chromium,having formed a solid precipitate, settles from the solution. Thetreated water 50, which has been substantially cleared of reducedchromium, can then be returned to the environment, for example by beingpumped into the soil. The waste product containing the precipitatedchromium can be further treated or otherwise disposed of in a mannerknown to one of ordinary skill in the art.

Versions of a treatment tank of the type which could be used in FIG. 10are shown in FIGS. 11 and 12. A packed-bed reactor is designated by thegeneral reference numeral 52 in FIG. 11. Contained within the reactor isa high-surface area packed-bed 54 which comprises an inert substrate,such as glass, plastic beads, or activated charcoal, upon which thebacteria can grow.

After a bacterial culture has been established on the packed-bed 54,contaminated water 56 and a nutrient medium 58 are introduced into andcirculated within the reactor 52. As noted above, growth conditions ofthe bacteria can be regulated. As the contaminated water percolatesthrough the packed-bed 54, bacteria act to reduce the Cr(VI). Treatedwater 59 leaves the reactor 52 and is introduced into a settling tank 48as described above wherein the precipitated chromium can be removed. Theresulting chromium waste product comprises a waste product orders ofmagnitude less in volume than the original water introduced into thesystem for treatment.

Shown in FIG. 12 is a fluidized-bead reactor designated by the generalreference numeral 60. The fluidized bed comprises a substrate 62 uponwhich the bacteria can grow as explained above. In this system thecontaminated water 64, and optionally nutrient medium 65, is forciblycirculated throughout the bed 62. This circulation process enhances theinteraction between the contaminated water and the bacteria upon the bed62. Treated water 66 flows from one outlet of the system. Nutrients areperiodically or continuously introduced into the system to maintainbacterial growth within the reactor 60. As precipitated chromium 68settles in the sump portion 69 of the reactor 60, it is collected in acollector 70 and removed for further treatment or disposed.

Either of the treatment systems described in FIGS. 11 or 12 could beused in the on-site treatment of industrial waste streams. In anotherembodiment of the invention, the contaminated soil 40 shown in FIG. 10could be inoculated in situ for reducing Cr(VI) contamination of theground water which comes into contact with it as described above.

All publications, references and patents referred to herein are herebyincorporated herein by reference.

Changes may be made in the construction and the operation of the variouscomponents, elements and assemblies described herein or in the steps orthe sequence of steps of the methods described herein without departingfrom the spirit and scope of the invention as defined in the followingclaims.

What is claimed is:
 1. A biologically pure culture of a Shewanella algastrain resistant to Chromium (VI) under anaerobic growth which isdeposited under ATCC accession number 55627.